S287: FACTORS INFLUENCING AUTOLOGOUS LYMPHOCYTE COLLECTIONS FOR CHIMERIC ANTIGEN RECEPTOR (CAR) T-CELLS – THE ROLE OF T-CELL SENESCENCE
Authors: V. Vucinic, T. Tumewu, M. Brückner, M. Jentzsch, F. Ramdohr, R. Buhmann, Y. Remane, S. Hoffmann, M. Janz, O. Penack, G. Vogtmann, E. Ruschpler, L. Bullinger, U. Keller, M. Cross, S. Schwind, M. Herling, G.-N. Franke, E. Bach, H. Reinhard, U. Platzbecker
Journal: HemaSphere (2022)

Background: The apheresis of autologous CD3+ lymphocytes is the first pivotal step in the production process of chimeric antigen receptor T-cells. A range of factors like previous cytotoxic therapies or disease status can influence the quality of collection but may also impact the fitness of T-lymphocytes. Repeated T-cell activation leads to progressive loss of expression of CD27 and CD28, receptors shown to be uniformly present on naïve CD4+ cells (van Leewen et al, J. Immunol, 2004). Aims: To evaluate factors influencing both collection of autologous lymphocytes and the subsequent manufacturing of tisagenlecleucel with a special focus on T-cell senescence, defined as loss of CD27 and CD28. Methods: Between February 2019 and October 2021, 59 collections were performed for subsequent CAR-T cell therapy with tisagenlecleucel in 51 patients with relapse/refractory (r/r) diffuse large B-cell lymphoma and one patient with refractory acute lymphoblastic leukemia. The median age was 60.5 (range 17-80) years and 40 (77%) patients were male. The collections were performed on a Spectra Optia cell-separator by processing a median 3x total blood volume. Results: The target numbers of CD3+ cells (>0.55 x109) were successfully collected within a single collection day in all patients but one. The median yield of CD3+ cells was 5.0 (0.4-31.9 x109, with yields for CD3+CD4+ and CD3+C8 + 20.1 (range 2.4-102.8) x108 and 27.4 (range 2.41-266.8) x108, respectively. The median CD4:CD8 ratio was 0.7 (0.08-5.3). The yields for CD3+CD27+CD28+ and CD3+CD27-CD28- cells were 25.9 (range 1.1-93.0) x108 and 10.9 (range 0.2-183.8) x108, respectively. We observed no difference in median collected CD3+ cell yields in patients with > vs ≤ 3 prior therapy lines (p=0.29), with or without prior treatment with bendamustine (p=0.42) or bone marrow infiltration (p=0.95). 56 collections underwent further manufacturing, with production according to specification in 38 (68%) collections. 18 collections resulted in production failure, 13 (72%) due to insufficient proliferation of T-cells, 1 (6%) because of microbiological contamination and 4 (22%) due to undeterminable test results for mycoplasms. The manufacturing failures did not associate with age >60 (p=0.16), sex (p=0.99), bone marrow infiltration (p=0.47) or >3 prior treatment lines (p=0.78). However, we did notice a trend regarding prior treatment with bendamustine (p=0.07). Importantly, the collections resulting in production failure had significantly lower CD3+/CD27+/CD28+ counts of 16.1 (range 1.1-52.3) vs 33.2 (range 3.3-93.5) x108 (p<0.01), as well as a trend for lower CD3+/CD4+ counts with 15.9 (range 3.0-43.9) vs 24.5 (range 2.4-102.7) x108 (p=0.05). However, there were no differences in CD3+/CD8+ counts (p=0.19), CD4:CD8 ratio (p= 0.77) or CD3+/CD27-/CD28- cells (p=0.81; Figure 1). Image:Summary/Conclusion: In our real-life cohort of lymphoma patients intended to undergo treatment with tisagenlecleucel, adequate numbers of CD3 cells could be collected in most cases irrespective of the number of prior therapies. We could observe a negative prognostic role of T-cell senescence on the manufacturing process. Further analyses will be required to determine measures appropriate for further optimization and improvement of collection outcomes.